The expression of bone matrix proteins induced by different bioimplants in a rabbit sinus lift model

This study aimed to analyze the expression of bone matrix proteins and CD31 by immunohistochemistry after maxillary sinus grafting with different bioimplants in a rabbit model. Rabbit demineralized bone matrix (DBM), partially purified bovine bone morphogenetic proteins (BMP), a mixture of BMP with DBM (BMP/DBM), or particulated autogenous bone was grafted into the maxillary sinuses of 42 rabbits. Animals were sacrificed at 2 and 8 weeks. Immunohistochemistry was used to investigate the expression of type 1 collagen (COL1), osteonectin (ON), osteocalcin (OC), bone sialoprotein (BSP), osteopontin (OPN), and CD31. Sinuses grafted with BMP were filled with trabeculae of woven bone that was strongly immunoreactive for COL1, OC, ON, and BSP. BMP/DBM showed strongly positive immunoreactivity for these proteins within the newly formed bone, but weak immunoreactivity in the DBM particles. Immunoreactivity for COL1, OC, ON, and BSP in DBM sinuses was only seen in the osteoblasts rimming the grafted bone particles. The staining of autogenous bone graft sinuses was similar to those grafted with DBM. OPN staining was detected in autogenous bone graft, BMP/DBM, and BMP bioimplants. CD31 staining was strongest in BMP and BMP/noncollagenous matrix proteins sinuses. These results suggest that exogenous BMP enhances not only osteogenesis but also angiogenesis, an important part of bone repair.     

Proliferation and osteoblastic differentiation of human bone marrow-derived stromal cells on akermanite-bioactive ceramics

In the present study, the effects of a calcium magnesium silicate bioactive ceramic (akermanite) on proliferation and osteoblastic differentiation of human bone marrow stromal cells (hBMSC) have been investigated and compared with the classical ceramic (beta-tricalcium phosphate, beta-TCP). Akermanite and beta-TCP disks were seeded with hBMSC and kept in growth medium or osteogenic medium for 10 days. Proliferation and osteoblastic differentiation were evaluated on day 1, 4, 7 and 10. The data from the Alamar Blue assay and lactic acid production assay showed that hBMSC proliferated more significantly on akermanite than on beta-TCP. The analysis of osteoblast-related genes, including alkaline phosphatase (ALP), osteopontin (OPN), bone sialoprotein (BSP) and osteocalcin (OC), indicated that akermanite ceramics enhanced the expression of osteoblast-related genes, but type I collagen (COL I) showed no noticeable difference among akermanite and beta-TCP ceramics. Furthermore, this stimulatory effect was observed not only in osteogenic medium, but also in normal growth medium without osteogenic reagents such as l-ascorbic acid, glycerophosphate and dexamethasone. This result suggests that akermanite can promote osteoblastic differentiation of hBMSC in vitro even without osteogenic reagents, and may be used as a bioactive material for bone regeneration and tissue engineering applications.     

Modified aminosilane substrates to evaluate osteoblast attachment, growth, and gene expression in vitro

Bone cell-substrate interactions are important to understand in the design, selection, and surface modification of bone implants. To gain insight into such interactions, substrates designed with surface species approximating the physiological environment of bone matrix were studied. Osteoblasts (Ob) grown on three such surfaces were used to evaluate cell-substrate effects on attachment, growth, and gene expression as compared with controls. Initial surface preparation consisted of coating glass slides with aminopropyltriethoxy silane (APTES), after which the coated slides were modified with collagen-rich extracellular matrix components obtained from normally mineralizing avian tendon: the tripeptide arginine-glycine-aspartic acid (arg-gly-asp), or a precipitate formed from a metastable solution containing inorganic ions normally found in blood (simulated body fluid). Each of the modified substrates, as well as the nonmodified (APTES) control, provided distinctly different physical (evidenced by differences in rms roughness) and chemical surfaces for seeding primary osteoblasts obtained from 14-day-old normal embryonic chickens. Cell responses to each of the substrates were evaluated over a 21-day period in terms of Ob growth and growth rate, alkaline phosphatase (ALP) activity, and gene expression of type I collagen (COL I), osteopontin (OPN), osteocalcin (OC), and bone sialoprotein (BSP). From these preliminary experiments, indications are that cell attachment and growth in this study possibly are independent processes, an assumption that compels the need for further studies. Collagen-rich matrix-modified substrates had a distinct advantage over others when cell growth rate, ALP activity, and gene expression were considered; cells on these substrates exhibited increased ALP activity and enhanced expression of BSP, OPN, and OC when compared with those of cells on APTES controls or other modified substrates. These results indicate that matrix-modified substrates such as those used in this study provide favorable templates for tissue generation, suggesting their potential in the design of surfaces for bone implants.     

In vitro generation of osteochondral differentiation of human marrow mesenchymal stem cells in novel collagen-hydroxyapatite layered scaffolds

Integrated, layered osteochondral (OC) composite materials and/or engineered OC grafts are considered as promising strategies for the treatment of OC damage. A novel biomimetic collagen-hydroxyapatite (COL-HA) OC scaffold with different integrated layers has been generated by freeze-drying. The capacity of the upper COL layer and the lower COL/HA layer to promote the growth and differentiation of human mesenchymal stem cells (hMSCs) into chondrocytes and osteoblasts respectively was evaluated. Cell viability and proliferation on COL and COL/HA scaffolds were assessed by the MTT test. The chondrogenic differentiation of hMSCs on both scaffolds was evaluated by glucosaminoglycan (GAG) quantification, alcian blue staining, type II collagen immunocytochemistry assay and real-time polymerase chain reaction in chondrogenic medium for 21 days. Osteogenic differentiation was evaluated by alkaline phosphatase activity assay, type I collagen immunocytochemistry staining, alizarin S staining and mRNA expression of osteogenic gene for 14 days in osteogenic medium. The results indicated that hMSCs on both COL and COL/HA scaffolds were viable and able to proliferate over time. The COL layer was more efficient in inducing hMSC chondrogenic differentiation than the COL/HA layer, while the COL/HA layer possessed the superiority on promoting hMSC osteogenic induction over either COL layer or pure HA. In conclusion, the layered OC composite materials can effectively promote cartilage and bone tissue generation in vitro and are potentially usable for OC tissue engineering.     

The effect of glow discharge plasma surface modification of polymers on the osteogenic differentiation of committed human mesenchymal stem cells

Little is known of the effect of material surfaces on stem cell differentiation. The present study has addressed the hypothesis that the interaction of mesenchymal stem cells (MSCs) with material surfaces modified by glow discharge plasma is a major regulator of osteogenic differentiation. We found that biaxially oriented polypropylene (BOPP) plasma treated in ammonia significantly reduced up-regulation of expression of osteogenic marker genes, such as alkaline phosphatase (ALP), bone sialoprotein (BSP) and osteocalcin (OC). In contrast, ALP expression was up-regulated when cultured on treated Nylon-6 polyamide (Ny-t) but was substantially reduced when cultured on its pristine counterpart (Ny-p) on day 3. On day 7, ALP expression was down-regulated with MSCs cultured on Ny-t although its expression level was up again on day 14. BSP was expressed weakly on day 3, but was up-regulated when cultured on Ny-t and Ny-p. Its expression reached its maximum on day 14 when cultured on a polystyrene control, while it was cyclically up-regulated on Ny-t. Similarly, there was a slight increase in OC expression when MSCs were cultured on Ny-t and Ny-p on day 3, when compared to control. Thus, the nature of the surface can directly influence MSCs differentiation, ultimately affecting the quality of new tissue formation with BOPP-t suppressing osteogenic differentiation.     

Interleukin-11 induces osteoblast differentiation and acts synergistically with bone morphogenetic protein-2 in C3H10T1/2 cells.

Interleukin-11 (IL-11) is a pleiotropic cytokine that supports various types of hematopoietic cell growth and is involved in bone resorption. We report here the involvement of recombinant human IL-11 (rHuIL-11) in osteoblast differentiation in mouse mesenchymal progenitor cells, C3H10T1/2. rHuIL-11 alone increased alkaline phosphatase (ALP) activity and upregulated expression levels of osteocalcin (OC), bone sialo protein (BSP), and parathyroid hormone receptor (PTHR) mRNA. rHuIL-11 had no effect on expression of type II collagen, peroxisome proliferator-activated receptor-gamma2 (PPAR-gamma2), adipocyte fatty acid-binding protein P2 (aP2), and myogenic MyoD protein (MyoD). Recombinant human bone morphogenetic protein (rHuBMP)-2 increased ALP activity and mRNA expression of these genes except for MyoD. The expression patterns of ALP activity and osteoblast-specific or chondrocyte-specific genes suggest that rHuIL-11 may be involved in early differentiation of osteoblasts at a step earlier than that which is affected by rHuBMP-2. In support of this hypothesis, combined treatment with rHuIL-11 and rHuBMP-2 synergistically increased ALP activity and mRNA expression of OC and type II collagen, rHuIL-11 also abrogated the increased levels of PPAR-gamma2, aP2 mRNA caused by rHuBMP-2. Our results suggest that rHuIL-11 alone and in combination with rHuBMP-2 can induce osteoblastic differentiation of progenitor cells and plays an important role in osteogenesis.     

Electroporation-mediated transfer of Runx2 and Osterix genes to enhance osteogenesis of adipose stem cells

In the present study, we tested the hypothesis that electroporation-mediated transfer of Runx2, Osterix, or both genes enhances the in vitro and in vivo osteogenesis from adipose stem cells (ASCs). ASCs were transfected with Runx2, Osterix, or both genes using electroporation, and further cultured in monolayer or in PLGA scaffold under osteogenic medium for 14 days, then analyzed for in vitro osteogenic differentiation. Transfected ASC-PLGA scaffold hybrids were also implanted on nude mice to test for in vivo ectopic bone formation. Runx2 and Osterix genes were strongly expressed in ASCs transfected with each gene on day 7, decreasing rapidly on day 14. Runx2 protein was strongly expressed in ASCs transfected with the Runx2 gene, while Osterix protein was strongly expressed in ASCs transfected with either or both Runx2 and Osterix genes. Overexpression of Runx2 and Osterix significantly increased the gene expression of osteogenic differentiation markers (alkaline phosphatase [ALP], osteocalcin [OCN], type I collagen [COL1A1], and bone sialoprotein [BSP]) in ASCs. Transfection of Runx2 and Osterix genes enhanced the protein expression of OCN, type I collagen, and BSP, as demonstrated by Western blot analysis, and ALP activity as well as enhancing mineralization in the monolayer culture and ASC-PLGA scaffold hybrids. Runx2- or Osterix-transfected ASC-PLGA scaffold hybrids promoted bone formation in nude mice after 6 weeks of in vivo implantation.     

Effects of Polygonatum sibiricum polysaccharide on the osteogenic differentiation of bone mesenchymal stem cells in mice

Polygonatum sibiricum polysaccharide (PSP) is a traditional Chinese medicine and is widely used to treat many diseases for hundreds of years conventionally. This study was to access the effects of PSP on the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in the mice. Cells collected from BALB/C mice in the bone marrow were isolated and cultured with osteogenic medium (OM) with different concentrations of PSP. The proliferation and morphological changes of BMSCs were observed using an inverted microscope. Flow cytometric analysis was used to identify the BMSCs. MTT test was performed to analyze the proliferation and viability of the cells. ELISA was used to determine the expression levels of alkaline phosphatase (ALP), osteocalcin (OC), N-terminal propeptide of type I procollagen (PINP) and bone morphogenetic protein-2 (BMP-2). Immunocytochemistry and western blot were respectively used to determine the expressions of bone sialoprotein (BSP) and SPARC/osteonectin (OSN). The growth curves of the proliferation and differentiation of the Control, OM, 17β-E₂ and PSP groups were increased. Compared to the Control and OM groups, the expression levels of ALP, OC, PINP and BMP-2 were significantly increased in the PSP induced group (P<0.05). Immunocytochemistry and western blot showed that BSP and SPARC were increased after induction of PSP compared to the OM group (P<0.05). The study demonstrates that PSP promotes the proliferation and enhances the viability of BMSCs during osteogenic differentiation. Therefore, PSP may be a potential treatment of osteoporosis in the clinic.     

Evaluation of human mesenchymal stem cells response to biomimetic bioglass-collagen-hyaluronic acid-phosphatidylserine composite scaffolds for bone tissue engineering

The aim of this study was to examine in vitro the response of human mesenchymal stem cells (hMSCs) on the novel biomimetic bioglass-collagen-hyaluronic acid-phosphatidylserine (BG-COL-HYA-PS) composite scaffold for potential use in bone tissue engineering. The initial attachment, the proliferation, migration and differentiation behavior of the cells on the BG-COL-HYA-PS composites were assessed in comparison with those on pure 58sBG, BG-COL, and BG-COL-HYA composites in either growth medium (L-DMEM supplemented with 10% fetal bovine serum) or osteogenic medium (growth medium supplemented with 0.1 microM dexamethasone, 10 mM beta-glycerophosphate, and 50 microM ascorbic acid). HMSCs attached, and subsequently proliferated and migrated on the BG-COL-HYA-PS composites to a significantly higher degree. The alkaline phosphatase (ALP) staining, ALP activity and the expression of the bone associated gene ALP, osteocalcin (OC), and osteopontin (OPN) was also significantly higher in the hMSCs on the BG-COL-HYA-PS scaffolds than those on the BG-COL, BG-COL-HYA composites and the pure 58sBG. These findings suggest that the BG-COL-HYA-PS composite porous scaffolds have high potential for use as scaffolds in bone tissue engineering and repair.     

Human osteoblast response to silicon-substituted hydroxyapatite

Human osteoblasts were cultured on hydroxyapatite (HA), 0.8 wt % silicon substituted hydroxyapatite (Si-HA) and 1.5 wt % Si-HA discs. The influence of these substrates on cell behaviour in vitro was assessed by measuring total protein in the cell lysate and the production of several phenotypic markers: collagen type I (COL I), alkaline phosphatase (ALP), osteocalcin (OC), and the formation of bone mineral. After 7 days, beta-glycerophosphate and physiological levels of hydrocortisone were added to the culture medium to stimulate cell differentiation and mineral production. There was a significantly higher production of ALP on 1.5 wt % Si-HA at day 7 following which, the addition of hydrocortisone promoted the differentiation of cells on the other two substrates. Hydrocortisone addition also decreased the production of OC. During the period, when hydrocortisone was present, no significant difference in behavior was seen between cells on Si-HA and HA; however, following removal of hydrocortisone, cells responded to 0.8 wt % Si-HA with a significant increase in protein production. Using fluorescence microscopy, nodular structures labeled with tetracycline were observed on the surface of all substrates after 21 days. These structures were deposited on areas of high cell density but were not related to the presence or level of silicon in the substrate. These results indicate that human osteoblasts are affected by the presence of silicon in the HA substrate and that the timing of these effects may be dependent upon the level of silicon substitution.     

In vitro and in vivo evaluation of akermanite bioceramics for bone regeneration

This study investigated the effects of a calcium magnesium silicate bioceramic (akermanite) for bone regeneration in vitro and in vivo, with β-tricalcium phosphate (β-TCP) as a control. In vitro, the human bone marrow-derived mesenchymal stromal cells (hBMSCs) were cultured in an osteogenic medium supplemented with a certain concentration of two bioceramics' extracts for 20 days. An MTT assay showed that akermanite extract promoted proliferation of hBMSC significantly more than did β-TCP extract. The results of alkaline phosphatase (ALP) activity test and the expression of osteogenic marker genes such as ALP, osteopontin (OPN), osteocalcin (OCN) and bone sialoprotein (BSP) demonstrated that the osteogenic differentiation of hBMSC was enhanced more by akermanite extract than by β-TCP extract. In vivo, a histomorphology analysis and histomorphometry of the two porous bioceramics implants in rabbit femur defect models indicated that both in early- and late-stage implantations, akermanite promoted more osteogenesis and biodegradation than did β-TCP; and in late-stage implantations, the rate of new bone formation was faster in akermanite than in β-TCP. These results suggest that akermanite might be a potential and attractive bioceramic for tissue engineering.     

The effect of heparin-functionalized PEG hydrogels on three-dimensional human mesenchymal stem cell osteogenic differentiation

Poly(ethylene glycol) (PEG) hydrogels functionalized with heparin were utilized as a three-dimensional culture system for human mesenchymal stem cells (hMSCs). Heparin-functionalized hydrogels supported hMSC viability, as quantified through live/dead imaging, and induced osteogenic differentiation, as measured by increased alkaline phosphatase (ALP) production and osteopontin (OPN) and collagen I (COL I) gene expression over the 5-week study. Further exploration of the potential mechanism of heparin-induced osteogenic differentiation was performed. Specifically, the availability of bone morphogenetic protein 2 (BMP2) and fibronectin (FN) in the culture system was controlled and hMSC osteogenic differentiation was evaluated as a function of the microenvironment. BMP2 availability increased both ALP production and OPN gene expression, while FN increased ALP production, but not OPN gene expression. Furthermore, immunostaining of integrin expression revealed that viability and differentiation were differentially affected by integrin production, where both alpha5beta1 and alphavbeta3 integrin-ligand interactions supported viability, while only the alpha5beta1 integrin played a role in hMSC osteogenic differentiation.     

Basic fibroblast growth factor enhances in vitro mineralization of rat bone marrow stromal cells grown on non-woven hyaluronic acid based polymer scaffold.

A biodegradable non-woven hyaluronic acid polymer scaffold (Hyaff 11) was analysed in vitro as a carrier vehicle for differentiation and mineralization of rat bone marrow stromal cells (BMSC). BMSC were grown on Hyaff 11 in a mineralizing medium in the presence/absence of basic fibroblast growth factor (bFGF). Osteoblastic differentiation was investigated by light and electron microscopy analysing the expression of osteogenic markers: calcium, alkaline phosphatase (AP), osteopontin (OP), bone sialoprotein (BSP) and collagen type 1. We also measured proliferation, AP activity and mRNA expression of AP and osteocalcin (OC). Electron microscopy and Toluidine-blue staining demonstrated that bFGF accelerated (day 20 vs. day 40) and increased mineralization. With bFGF, calcium, OP and BSP were strongly enhanced at day 40, whereas AP decreased. Our in vitro results demonstrate that Hyaff 11 is a useful vehicle for growth, differentiation and mineralization of rat BMSC, and that it permits bone development.     

The expression of bone matrix protein mRNAs around beta-TCP particles implanted into bone

Tissue response around beta-tricalcium phosphate (beta-TCP) particles (150-300 microm in diameter) implanted into rat tibiae was analyzed by in situ hybridization with digoxigenin-labeled procollagen alpha1(I) (COL), osteonectin, osteocalcin, and osteopontin (OPN) RNA probes. Specimens were collected at 3, 5, 7, and 10 days after the operation. Holes without implantation were used as control. In both the beta-TCP implanted and control groups, new bone was formed centripetally and all four kinds of mRNA were expressed in activated osteoblasts. A COL signal was expressed most strongly and widely, and was detected at the peripheral region of the hole at day 3. The other three mRNAs were also expressed in bone forming osteoblasts by day 7. However, in the earlier cell reaction stage, OPN expression in the beta-TCP implanted group was different than that in the control group: OPN mRNA was seen exclusively in the cells on the particles, and an OPN signal was detected not only in COL-positive cells, but also in COL-negative cells. The former cells may be osteoblasts and reflect the early process of bone formation on biomaterials. The latter cells may be macrophages and reflect foreign body reactions. Expression of these OPN mRNAs induced by implantation of beta-TCP may play a role in bone formation on the materials and in determining their biocompatibility.     

Effect of surface chemical modification of bioceramic on phenotype of human bone-derived cells.

In the search for methods to improve the biocompatibility of prosthetic materials, attention has recently been directed toward the potential use of surface chemical modification and its influence on cellular behavior. This in vitro study investigates the effect of surface chemistry modification of bioceramics on human bone-derived cells (HBDCs) grown on biomaterial surfaces for 2 weeks. Cells were cultured on either alumina (Al2O3), alumina doped with magnesium ions ([Mg]-Al2O3), or hydroxyapatite (HAP), as well as tissue culture polystyrene (TCPS). Expression of alkaline phosphatase (ALP), thrombospondin (Tsp), osteopontin (OP), osteocalcin (OC), osteonectin (ON/SPARC), type I collagen (Col I), and bone sialoprotein (BSP) were determined in terms of mRNAs and proteins. Protein levels for ALP, OP, OC, and BSP were significantly (p < 0. 05) greater at day 5 in HBDCs cultured on [Mg]-Al2O3 compared to those cells grown on Al2O3. At day 14 the levels of ALP, Tsp, Col I, OP, ON/SPARC, and BSP rose significantly (p < 0.05) above those occurring in HBDCs grown on Al2O3, HAP, and TCPS. This suggests that HBDCs from the same patient respond to differences in the surface chemical groups. This study confirms that the chemistry of a substratum, which facilitates cellular adhesion, will enhance cellular differentiation.     

In vivo bone formation following transplantation of human adipose-derived stromal cells that are not differentiated osteogenically

A number of studies have shown in vivo bone regeneration by transplantation of osteogenic cells differentiated in vitro from adipose-derived stromal cells (ADSCs). However, the in vitro osteogenic differentiation process requires an additional culture period, and the dexamethasone that is generally used in the process may be cytotoxic. Here, we tested the hypothesis that ADSCs that are not differentiated osteogenically in vitro prior to transplantation would extensively regenerate bone in vivo when exogenous bone morphogenetic protein-2 (BMP-2) is delivered to the transplantation site. We fabricated a poly(dl-lactic-co-glycolic acid)/hydroxyapatite (PLGA/HA) composite scaffold with osteoactive HA that is highly exposed on the scaffold surface. This scaffold was able to release BMP-2 over a 4-week period in vitro. Human ADSCs cultured on BMP-2-loaded PLGA/HA scaffolds for 2 weeks differentiated toward osteogenic cells expressing alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) mRNA, while cells on PLGA/HA scaffolds without BMP-2 expressed only ALP. To study in vivo bone formation, PLGA/HA scaffolds (group 1), BMP-2-loaded PLGA/HA scaffolds (group 2), undifferentiated ADSCs seeded on PLGA/HA scaffolds (group 3), and undifferentiated ADSCs seeded on BMP-2-loaded PLGA/HA scaffolds (group 4) were implanted into dorsal, subcutaneous spaces of athymic mice. Eight weeks after implantation, group 4 exhibited a 25-fold greater bone formation area and 5-fold higher calcium deposition than group 3. Bone regeneration by transplanted human ADSCs in group 4 was confirmed by expression of human-specific osteoblastic genes, ALP, collagen type I, OPN, OCN, and bone sialoprotein, while group 3 expressed much lower levels of collagen type I and OPN mRNA only. This study demonstrates the feasibility of extensive in vivo bone regeneration by transplantation of ADSCs without prior in vitro osteogenic differentiation, and that a PLGA/HA composite BMP-2 delivery system stimulates bone regeneration following transplantation of undifferentiated human ADSCs.     

Tissue responses around polymethylmethacrylate particles implanted into bone: analysis of expression of bone matrix protein mRNAs by in situ hybridization.

Tissue responses around implanted polymethylmethacrylate (PMMA) particles were analyzed by in situ hybridization with digoxigenin-labeled procollagen alpha1(I) (COL), osteonectin, osteocalcin, and osteopontin (OPN) mRNA probes. PMMA particles (150-300 microm in diameter) were implanted into rat tibiae, and specimens were collected at 3, 5, 7, and 10 days after operation. New bone was formed centripetally, and bone-forming osteoblasts expressed all four kinds of mRNAs. A COL signal was expressed most strongly and widely. In the early stage, COL-positive cells were detected on and among particles sporadically. A COL signal was rarely detected in cells on the surfaces of the particles, suggesting that PMMA particles may suppress osteoblast differentiation. Osteonectin and osteocalcin mRNAs were expressed in bone-forming osteoblasts in a similar pattern by day 7. By contrast, an OPN signal was detected mainly on the particles, not only in COL-positive osteoblasts but also in COL-negative round cells. The latter cells had acid phosphatase activity, suggesting that they might be macrophages responding to a foreign body. At day 10, an OPN signal was detected continuously in multinucleated cells on PMMA particles, whereas new bone was formed away from particles. Our approach helped us to understand the initial cellular reaction to materials, which may determine their biocompatibility.